We study the mechanism and regulation of protein synthesis in eukaryotic cells with focuses on regulation by GTP-binding (G) proteins and protein phosphorylation as well as unusual post-translational modifications of the factors that assist the ribosome in synthesizing proteins. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is a G protein and during the course of translation initiation the GTP is hydrolyzed to GDP. eIF2 is released from the ribosome in complex with GDP and the guanine-nucleotide exchange factor eIF2B converts eIF2-GDP to eIF2-GTP. Phosphorylation of the alpha subunit of eIF2 on serine 51 coverts eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2 (activated under conditions of amino acid starvation), PKR (activated by double-stranded RNA to down-regulate protein synthesis in virally infected cells), PERK (activated under ER stress conditions), and HRI (activated under conditions of low heme). eIF2 is composed of three subunits. The gamma subunit of eIF2 is a GTPase that resembles the bacterial translation elongation factor EF-Tu. We previously showed that despite their structural similarity eIF2 and EF-Tu bind tRNA in substantially different manners, and we showed that the tRNA-binding domain III of EF-Tu has acquired a new function in eIF2gamma to bind the ribosome. As described below, our structure-function studies on eIF2 have provided insights into human disease. Whereas protein synthesis plays a critical role in learning and memory in model systems, human intellectual disability syndromes have not been directly associated with alterations in protein synthesis. Working with collaborators in Israel and Germany, we characterized a human X-linked disorder characterized by intellectual disability and microcephaly. The patients carry a mutation in the EIF2S3 gene encoding eIF2gamma, and genetic and biochemical studies revealed that the mutation disrupts eIF2 complex integrity and translation start codon selection. These findings directly link intellectual disability to impaired translation initiation, and provide a mechanistic basis for the human disease due to partial loss of eIF2 function. Over the past year, working with additional collaborators, we are characterizing new mutations in eIF2gamma that cause intellectual disability and we have linked these mutations to MEHMO syndrome. When expressed in yeast, human PKR phosphorylates the alpha subunit of eIF2 on Ser51 causing inhibition of protein synthesis and yeast cell growth. To subvert the anti-viral defense mediated by PKR, viruses produce inhibitors of the kinase. We are currently characterizing mutations in PKR that confer resistance to the poxviral E3L inhibitor. In a related project, we are characterizing the insect baculovirus PK2 protein, an eIF2alpha kinase inhibitor that structurally mimics the C-terminal lobe of a protein kinase domain. Together with collaborators in Canada and Japan, we characterized PK2 mutants, revealed that PK2 associates with the N-lobe of eIF2alpha kinases, and identified an insect HRI-like kinase as the likely target of PK2. Consistent with this notion, our collaborators showed that knockdown of the HRI-like kinase in insects rescued viral defects associated with loss of PK2. We propose an inhibitory mechanism whereby PK2 engages the N-lobe of an eIF2alpha kinase domain to create a nonfunctional pseudokinase domain complex, possibly through a lobe-swapping mechanism. In our final two projects we are studying the translation factors eIF5A and eEF2. These proteins both function in translation elongation and interestingly carry novel post-translational modifications. eIF5A is the sole protein containing the unusual amino acid hypusine, N-epsilon-(4-amino-2-hydroxybutyl)lysine. The hypusine residue in eIF5A is found in all eukaryotes and archaea. Using molecular genetic and biochemical studies, we showed that eIF5A promotes translation elongation, and that this activity is dependent on the hypusine modification. We also showed that eIF5A from yeast, like its bacterial ortholog EF-P, stimulates the synthesis of proteins containing runs of consecutive proline residues. Consistent with these in vivo findings, we showed that eIF5A was critical for the synthesis of polyproline peptides in reconstituted yeast in vitro translation assays, and using directed hydroxyl radical probing experiments we mapped eIF5A binding near the E site of the ribosome. Over the last year, and working with x-ray crystallographers in France, we obtained a 3.25-angstrom resolution crystal structure of eIF5A bound to the yeast 80S ribosome. The structure reveals interactions between eIF5A and conserved ribosomal proteins and rRNA bases. Moreover, eIF5A occupies the E site with the hypusine residue projecting toward the acceptor stem of the P-site tRNA. Our studies reveal a previously unseen conformation of an eIF5A-ribosome complex, suggest a function for eIF5A and its hypusine residue to reposition the polyprolyl-tRNA in the P site to alleviate stalling, and highlight a possible functional link between conformational changes of the ribosome during protein synthesis and the eIF5A-ribosome association. Finally, together with collaborators at the MRC in Cambridge, we are studying the translation elongation factor eEF2. The eEF2, like its bacterial ortholog EF-G, promotes translocation of tRNAs and mRNA on the ribosome following peptide bond formation. In all eukaryotes and archaea, a conserved histidine residue at the tip of eEF2 is post-translationally modified to diphthamide through the action of 7 non-essential proteins. The function of diphthamide and rationale for its evolutionary conservation are not well understood, and to date the only known function of diphthamide is to serve as a substrate for inactivation by diphtheria toxin. To gain insights into the function of eEF2 and diphthamide, we reconstituted the function of the cricket paralysis virus (CrPV) internal ribosome entry site (IRES) in a yeast in vitro translation assay system. The CrPV IRES is unique in bypassing the requirement for any translation initiation factors. Thus, the IRES binds directly to the A-site of the ribosome. Following eEF2-directed pseudotranslocation of the IRES to the P site, an aminoacyl-tRNA binds to the A-site following by a second pseudotranslocation to move the IRES to the E site and the A-site tRNA to the P site. Following peptide bond formation, normal translation elongation ensues requiring the factors eEF1A and eEF2 and the yeast-specific factor eEF3. Using the canonical initiation pathway to direct the synthesis of the peptide Met-Phe-Lys revealed no distinction between eEF2 with or without the diphthamide modification. In contrast, synthesis of this same peptide directed by the CrPV IRES was sensitive to loss of diphthamide. As the pseudotranslocation steps are the main distinguishing feature of the CrPV IRES system, we propose that the precise phasing of pseudotranslocation is dependent on the diphthamide modification on eEF2. Consistent with this interpretation, using electron cryomicroscopy (cryo-EM) our collaborators revealed that eEF2 stabilizes the ribosome-IRES complex in a rotated state with key residues in domain IV of eEF2 interacting with the CrPV-IRES and stabilizing it in a conformation reminiscent of a hybrid tRNA state. Interestingly, diphthamide appears to break decoding interactions between conserved rRNA bases and the tRNA-mimicking pseudoknot I of the CrPV IRES. Thus, our studies provide the first evidence that diphthamide plays a role in protein synthesis, and we propose that it functions to disrupt the decoding interactions of rRNA in the A-site and to maintain codon-anticodon interactions as the A-site tRNA is translocated to the P site.